Bioartificial liver

ABSTRACT

This document provides bioartificial liver (BAL) devices. Methods for making and using BAL devices also are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a {continuation or continuation-in-part ordivisional} application of and claims priority to U.S. ProvisionalApplication Ser. No. 61/160,150, filed on Mar. 13, 2009.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under DK56733 awarded bythe National Institutes of Health. The government has certain rights inthe invention.

TECHNICAL FIELD

This document relates to bioartificial livers.

BACKGROUND

According to the annual report of the American Liver Foundation in 2000,hepatitis and other liver diseases affect 25 million Americans. Liverfailure is the 8th most frequent cause of death in the United States,accounting for roughly 43,000 deaths each year. Liver transplantation iscurrently the only effective treatment for medically refractory liverfailure. Liver transplantation has some shortfalls, however, including ashortage of donor organs, restrictions on potential recipients, and sideeffects of drugs used to prevent rejection after transplantation. Theproblem of organ scarcity is demonstrated by the fact that nearly 2000candidates for liver transplantation died on the waiting list in 2003.Over 500 of these patients were listed with a diagnosis of fulminanthepatic failure (FHF). One potential solution to the shortage of organsis an extracorporeal liver support system, or bioartificial liver (BAL),which could serve as a bridge to transplantation or, in some cases,until spontaneous recovery of the native liver.

One goal of BAL therapy is to provide detoxification activity andpreventing the systemic manifestations of acute liver injury asillustrated in FIG. 1. Current knowledge is that extrahepaticmanifestations of ALF, such as brain edema, lung dysfunction, and renaldysfunction, result from a two-hit process: 1) systemic inflammatoryresponse syndrome (SIRS); 2) occurring in the setting of inadequatehepatic synthetic and detoxification activities. The inflammatoryresponse of ALF is mediated by cytokines, such as TNFα, that can bereleased from the acutely injured liver, while toxins such as ammoniaapply a direct cytotoxic influence on target organs and their supportingcells. In support of this theory is the observation that SIRS is seen inat least 60% of ALF cases.

Ammonia, normally eliminated by the liver, is the most important toxinof the two-hit hypothesis outlined in FIG. 1. Arterial concentrations ofammonia have been shown to correlate with cerebral herniation inpediatric patients with urea cycle disorders and in many, but not all,ALF patients. TNFα is also an important marker of SIRS and the two-hithypothesis of ALF. Furthermore, cytokines such as TNFα are believed tomediate the local inflammatory response and brain edema of head trauma.Therefore, it is highly conceivable that a synergism exists betweeninflammation and ammonia resulting in extrahepatic manifestations ofALF, such as brain edema.

SUMMARY

This document provides spheroid reservoir bioartificial livers (SRBALs),methods of making SRBALs, and methods for using SRBALs. In some cases,the SRBALs described herein can include a multi-shelf rocking devicethat is configured to rock a plurality of cell containers for forminghepatocyte spheroids. In some cases, the SRBALs described herein caninclude a reservoir chamber for housing hepatocyte spheroids that has ascreen member to allow bi-directional fluid flow. In some cases, the ascreen member can be designed to prevent loss of hepatocyte spheroidsfrom the reservoir chamber. The screen member can be a membrane, afilter, or a mesh with openings in the micron range. In some cases, thereservoir chamber can include a fenestrated funnel-shaped settlingcolumn or cylindrical shaped settling column to prevent loss of spheroidhepatocytes into the outflow leaving the reservoir. In some cases, theSRBALs described herein can be liver support devices that combine livercell therapy with albumin dialysis to create a hybrid system thataddresses past limitations of both forms of extracorporeal artificialliver therapy. In such SRBALs, albumin dialysis and primary hepatocytescan be synergistic for treatment of ALF.

In general, one aspect of this document features a bioartificial liverdevice comprising a reservoir chamber configured to house hepatocytespheroids, wherein the reservoir chamber comprises a mixing chamber anda settling volume chamber, wherein the mixing chamber is separated fromthe settling volume chamber by a funnel, and wherein the mixing chamberis in fluid communication with the settling volume chamber via at leastone opening defined in the funnel. The funnel can be a fenestratedfunnel. The fenestrated funnel can comprise openings between 100 μm and5 mm in diameter. The reservoir chamber can comprise a magnetic stir barwithin the mixing chamber. The mixing chamber can comprise an inletport. The settling volume chamber can comprise an outlet port. Thefunnel can comprise more than 25 openings. The openings can be between100 μm and 5 mm in diameter. The reservoir chamber can comprise asampling port. The reservoir chamber can comprise temperature probe.

In another aspect, this document features a bioartificial liver devicethat includes a plurality of cell containers to form hepatocytespheroids. The device can also include a multi-shelf rocking device thatis configured to rock the plurality of cell containers. The device canfurther include a reservoir chamber that is configured to house thehepatocyte spheroids after they are formed in the plurality of cellcontainers.

In some embodiments, the bioartificial liver device can also include analbumin dialysis system. In some embodiments, the albumin dialysissystem can include a blood separation cartridge, a charcoal column, aresin column, and a dialysis membrane. In some embodiments, the albumindialysis system can include a pre-dilution circuit.

In some embodiments, the multi-shelf rocking device can be configured torock at about 5-20 cycles/min. In some embodiments, the multi-shelfrocking device can include one or more rocker boxes having a membranefor gas inflow/outflow.

In some embodiments, the reservoir chamber can include a mixing device(e.g., a spinning device or an impeller/propeller device) to maintainhepatocyte spheroids in suspension. In some embodiments, the reservoirchamber can be configured to allow bi-directional fluid flow.

In another aspect, this document features a bioartificial liver devicethat includes a reservoir chamber that is configured to house hepatocytespheroids and includes a membrane to allow bi-directional fluid flow. Insome embodiments, the membrane can have a pore size in the micron range(e.g., about 20-40 microns or smaller).

In some embodiments, the bioartificial liver device can also include analbumin dialysis system. In some embodiments, the albumin dialysissystem can include a blood separation cartridge, a charcoal column, aresin column, and a dialysis membrane. In some embodiments, the albumindialysis system can include a pre-dilution circuit.

In some embodiments, the bioartificial liver device can also include amulti-shelf rocking device that is configured to rock a plurality ofcell containers to form hepatocyte spheroids. In some embodiments, thereservoir chamber can also include a mixing device configured tomaintain hepatocyte spheroids in suspension. In some embodiments, thereservoir chamber can also include a gas permeable membrane tofacilitate gas exchange.

In another aspect, this document features a bioartificial liver devicethat includes an albumin dialysis system. The device can also include areservoir chamber that is in fluid communication with the albumindialysis system and is configured to house hepatocyte spheroids.

In some embodiments, the albumin dialysis system can include a bloodseparation cartridge, a charcoal column, a resin column, and a dialysismembrane. In some embodiments, the albumin dialysis system can include apre-dilution circuit.

In some embodiments, the bioartificial liver device can also include amulti-shelf rocking device that is configured to rock a plurality ofcell containers to form hepatocyte spheroids.

In some embodiments, the reservoir chamber can include a mixing deviceto maintain hepatocyte spheroids in suspension. In some embodiments, thereservoir chamber can include a screen (e.g., a membrane, a filter, or amesh with openings in the micron range) that is configured to allowbi-directional fluid flow or that is configured to prevent loss ofspheroid hepatocytes from the reservoir chamber. In some cases, thereservoir chamber can be designed to function as a settling column toprevent loss of hepatocyte spheroids. In some embodiments, the reservoirchamber can include a gas permeable membrane to facilitate gas exchange.In some cases, a screen or spacer can be located between the gaspermeable membrane and the inner wall of the reservoir to allow uniformgas flow through the device.

In some embodiment, the bioartificial liver device can also include acontroller to stabilize fluid volume in the reservoir chamber.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1: The two-hit hypothesis suggests that the systemic manifestationsof acute liver injury which may include cerebral edema, fever,hypotension, lung dysfunction, renal failure, and death result from asynergistic accumulation of toxins after loss of hepatocellular function(i.e., ammonia accumulation from loss of ureagenesis) and a systemicinflammatory response of unclear etiology but not likely to beinfectious in origin. Data indicates that the SRBAL prevents thesystemic manifestations of acute liver injury by correcting both arms ofthis process.

FIG. 2: Pig hepatocyte spheroids provide the metabolic anddetoxification activity to the SRBAL device. Examples of porcinehepatocyte spheroids are shown after 1 week in the rocked culturedsystem which is utilized in an exemplary SRBAL device.

FIG. 3: Schematic (a), photographic (b), and diagrammatic (c)illustrations of an exemplary SRBAL device for use in, for example,preclinical trials. The SRBAL is a “hybrid” extracorporeal liver supportdevice that includes an albumin dialysis system and a living biologicalcomponent. The hepatocyte reservoir can have spheroids of primaryporcine hepatocytes. The 5-pump SRBAL device places the hepatocytereservoir into the albumin perfusate of a standard albumin dialysisextracorporeal configuration. The albumin perfusate circuit includescharcoal and resin columns as currently configured in the MARS™ circuit(Gambro, Lund Sweden).

FIG. 4: Spheroid reservoir configurations: (a) rocker reservoir forspheroid formation; (b) multi-shelf rocker for large-scale spheroidformation; (c) spinner reservoir for extracorporeal use; and (d)schematic of spinner reservoir.

FIG. 5: Systemic Inflammatory Response Syndrome (SIRS) of D-Gal-InducedALF is associated with extrahepatic organ dysfunction—includingrespiratory failure as shown in (a). Using serum TNFα as a marker ofSIRS, the SIRS response of ALF was normalized by treatment with theHepatocyte SRBAL (b). Clearance of LPS by Kupffer cells of the liver wassuggested by the gradient between LPS levels sampled in blood from theportal vein and hepatic vein (c). The rise in TNFα activity after D-Galin dogs could not be explained fully by a short transient release of LPSfrom the gut, suggesting the existence of other initiators of SIRS innon-infectious acute liver failure.

FIG. 6( a-c): Role of TLR4 in signaling liver injury and SIRS aftertoxin exposure.

FIG. 7: (a) TLR4 Signaling Complex is found in many cell types. It canalso be stably transfected into reporter cells to measure TLR4 agonistactivity in vitro; (b) is a schematic representation of HEK293 cellswhich have been transfected with TLR4 complex and a luciferase reportergene to measure TLR4 agonist activity in vitro. TheHEK293/Luc(+)/TLR4(+) cells, used to measure TLR4 agonist activity inmouse serum in (c) are used to screen dog serum for TLR4 agonistactivity; (c) TLR4 agonist activity was elevated in mouse blood aftertoxic exposure to APAP (500 vs 0 mg/kg). This in vitro assay is based ona luciferase reporter gene linked to NFκβ in HEK293 cells which respondsto TLR4 activation. Only trace background activity was detected inHEK293 cells lacking the TLR4 complex, labeled TLR4(−), confirmingspecificity of the luciferase activity to TLR4.

FIG. 8: Comparison of rocked vs. rotational conditions (upper panel)along with hepatocyte spheroids viewed by phase microscopy duringspheroid formation (lower panel). Hepatocytes (1×10⁶ cells/mL) wererocked at 0.25 Hz or rotated at 60 rpm to induce spheroid formation.Representative images of spheroid formation by rocked technique areprovided at 4 hr, 12 hr, and 24 hr and by rotational technique at 4 hr,12 hr, and 24 hr. Scale bars equal 50 μm.

FIG. 9: Representative hepatocyte spheroids stained by Fluoroquench™after 5 days in rocked culture. Viable cells stained green while deadcells stained red. (a) low power of spheroids; (b) single spheroiddemonstrating numerous viable cells by FITC filter; (c) same spheroidwith a small focus of 2-3 dead cells by rhodamine (red) filter.

FIG. 10: Expression of 242 liver specific genes by a microarray inrocked spheroid culture over 14 days. Average expression of over 80% ofthese genes was stable over time (green color). A reduction in geneexpression from baseline (freshly isolated rat hepatocytes) is indicatedin pink or red, while an increase in expression is labeled blue.

FIG. 11: Schematic of bench-top apparatus used for testing flowconditions, medium conditions, and hollow fiber membranes underoptimization studies. Under diffusion conditions pumps were set so thatF=0 mL/min (P1=P2=P3=100 mL/min). Pumps were set so that F=5 mL/min, 25ml/min, or 50 mL/min under conditions of low, medium and high convectiveflux, respectively.

FIG. 12: Effect of rotational speed of spinner reservoir. (a) Effect ofimpeller speed on spheroid quantity and size distribution; (b)micrographs of spheroids after 22 hours of mixing; and (c) caspaseactivity normalized for spheroid volume. These figures show that a rapidspeed of spinning of 196 RPM vs. 98 PRM can have a favorable effect onforming spheroids of smaller size without the adverse effect of celldeath since caspase 3/7 activity and Fluoroquench™ vital staining ofhepatocytes is comparable at both rates of spinning.

FIG. 13: Formation of spheroids in both serum-containing and serum-freemedium. Spheroids form in both serum free medium and medium supplementedwith 10% FBS. L-carnatine can have a favorable effect on spheroidformation.

FIG. 14 (a-b): Diagrammatic (a) and photographic (b) illustration of anexemplary SRBAL device having four pumps. The SRBAL is a “hybrid”extracorporeal liver support device that includes an albumin dialysissystem and a living biological component. The hepatocyte reservoir canhave hepatocyte spheroids.

FIG. 15 (a-b): Diagrammatic (a) and photographic (b) illustration of anexemplary fenestrated funnel design of a spheroid reservoir.

FIG. 16: Diagrammatic illustration of an exemplary plain funnel designof a spheroid reservoir.

FIG. 17: Diagrammatic illustration of an exemplary straight settlingcolumn design of a spheroid reservoir.

FIG. 18: Table providing oxygen consumption results of pig hepatocytespheroids in culture in the multishelf rocker (Day 0, 1, 2) and thefenestrated funnel settling column reservoir (hours 0-24).

FIG. 19: Table providing data demonstrating stable cell volume duringspheroid formation under low pO₂ conditions.

FIG. 20: Table providing data demonstrating high yield of spheroids frompig hepatocytes under low pO₂ conditions.

FIG. 21: Graph plotting size distribution of microbead particles inreservoir and outflow line under spinner flask and fenestrated funnelconfigurations of the SRBAL bioreactor. Large particles were moreeffectively retained by the fenestrated funnel configuration.

DETAILED DESCRIPTION

A SRBAL provided herein is an extracorporeal artificial liver device. Insome cases, a SRBAL provided herein can include a multi-shelf rockingdevice for forming hepatocytes spheroids. In some cases, a SRBALprovided herein can include a screen or mesh with micron sized openingsthat allows bi-directional fluid flow. In some cases, the spheroidreservoir chamber can include a fenestrated funnel settling column toprevent loss of hepatocyte spheroids from the chamber. In some cases, aSRBAL provided herein can include an albumin dialysis system. Thealbumin dialysis system of the device can be analogous to the MARS®albumin dialysis system produced by Gambro, Inc (Lund, Sweden) with themodification of a larger pore size membrane (150 kD-400 kD) or smallerand addition of a Dialysis Hollow Fiber Filter for ultrafiltration,pre-dilution, and convective mass transfer across the Blood hollow fibermembrane filter. In some cases, a SRBAL provided herein can include aliving biological component (e.g., hepatocytes obtained from mammalianlivers including human, equine, canine, porcine, bovine, ovine, andmurine sources). For example, the living biological component can haveporcine hepatocytes in a 3-dimensional tissue construct (i.e., spheroid)as illustrated in FIG. 2. The spheroid aggregate design can be asdescribed in U.S. Pat. No. 7,160,719 to Nyberg. The spheroids of porcinehepatocytes can be isolated in a reservoir chamber located in continuitywith an albumin perfusate of the albumin dialysis system. An example ofa SRBAL device is illustrated schematically, photographically, anddiagrammatically in FIGS. 3 a, 3 b, and 3 c, respectively. Anotherexample of a SRBAL device provided herein is illustrateddiagrammatically and photographically in FIGS. 14 a and 14 b,respectively.

The SRBAL device can be designed for continuous extracorporeal therapyof patients with liver failure. The patient's blood circulation may beconnected to the device by a two-lumen venovenous catheter which is ableto achieve blood perfusion rates on the order of 100-200 mL/min. Thedevice can operate analogous to continuous venovenous hemodialysis. Inthis exemplary embodiment, the device contains five pumps as illustratedin FIG. 3( c) (pumps labeled PM1-5). Pumps 1 and 3 are used to perfusethe albumin circuit. Pump 2 perfuses blood from the patient to thehollow fiber blood separation cartridge. Pump 4 is used for discard ofultrafiltration fluid as needed to maintain the patient's fluidstatus—analogous to continuous venovenous hemodialysis. Pump 5 controlsa pre-dilution circuit used to achieve high ultrafiltration rates acrossthe hollow fiber membrane which is located at the intersection of thepatient blood circuit and the albumin circuit.

In some cases, the device can be designed to have four pumps asillustrated in FIG. 14 a (pumps labeled PM1-4). Pump 3 is used toperfuse the albumin circuit. Pump 2 perfuses blood from the patient tothe hollow fiber blood separation cartridge. Pump 4 is used for discardof ultrafiltration fluid as needed to maintain the patient's fluidstatus—analogous to continuous venovenous hemodialysis. Pump 1 controlsa pre-dilution circuit used to achieve high ultrafiltration rates acrossthe hollow fiber membrane which is located at the intersection of thepatient blood circuit and the albumin circuit. In some cases, the devicecan include an air/foam trap positioned above the spheroid reservoir asshown in FIG. 14 a.

Along with the spheroid reservoir, the albumin circuit includes acharcoal column, a resin column, a fluid warmer, and a second hollowfiber membrane module for ultrafiltration. The device can bemicroprocessor controlled to stabilize fluid volumes in the spheroidreservoir and accomplish ultrafiltration if desired for fluid removal.The device can include various controllers to regulate temperature, pHand/or O₂. The SRBAL device can provide multiple aspects ofextracorporeal liver support including detoxification of waste materialsfrom the patient and delivery of water metabolites and growth factorsand other, as yet unidentified products of primary liver cells back tothe patient. These processes may involve mass transport across thehollow fiber membrane by both diffusion and convection. Diffusion can beprimarily determined by the surface area of the hollow fiber membraneand the concentration gradient across the membrane, while convection canbe determined by the flow of fluid across the membrane set by pumpingconditions.

In some implementations, a dose of 200-400 g hepatocytes (e.g., porcinehepatocytes) is used in the SRBAL. The dose can be prepared by startingwith 5−10×10⁶ cells/mL in 6-12 liters of medium placed in a multi-shelfrocker device, then concentrating to put 1−2×10⁷ cells/mL (e.g.approximately 200-400 g) in a 3 L spheroid reservoir. In someimplementations, the albumin concentration may be in a range of 0.5 to5.0 g/dL. In some implementations, the albumin circuit can include apre-dilution feature such as a circuit off of the Dialysis Hollow FiberMembrane Filter to the inlet air/foam trap and involving pump 5 shown inFIG. 3 c. Pre-dilution may allow for convective flow across the membraneof the Blood Hollow Fiber Membrane filter without excessivehemoconcentration and sludging of blood in the patient's blood circuit.Increased mass transfer from the patient to the albumin circuit andhepatocyte reservoir can have the benefit of increased detoxification bythe SRBAL. In some implementations, pumps 1 and 3 (PM1 and PM3) as shownin FIG. 3 c are operated at the same rate—a rate that circulates fluidto and from the hepatocyte spheroid reservoir increase difussive masstransfer. In some implementations, pump 4 (PM4) as shown in FIG. 3 c maybe set to remove fluid based on a clinical need (e.g., renal replacementoutflow). In some implementations, pump 5 (PM5; recycle circuit) asshown in FIG. 3 c may be set to provide predilution and better masstransfer across the hollow fiber membrane via convective flow. The flowrate of pump 5 as shown in FIG. 3 c can determine how much ultrafiltratefluid from the albumin circuit is recycled to the inlet of the BloodHollow Fiber membrane filter for predilution while pump 4 as shown inFIG. 3 c determines how much fluid is removed from the patient. Thespheroid reservoir volume can be held constant during standard operatingconditions. Together, the rates of pump 4 and pump 5 can determine therate of convective flow (F) across the hollow fiber membrane. If pump 4and pump 5 are set at 0 mL/min, then there is no convection flow acrossthe hollow fiber membrane (F=0), and mass transport occurs by purediffusion. If pump 4 and/or pump 5 are in operation, then convectionoccurs across the hollow fiber membrane. Under standard conditions, theblood separation hollow fiber filter can have pores of 150 kD-400 kD.This pore size is larger than MARS™ (70 kD; Gambro, Lund Sweden).

In some cases, the device can have four pumps as shown in FIG. 14.

FIG. 4 a shows a rocker reservoir for spheroid formation. FIG. 4 b showsa multi-shelf rocker for large-scale spheroid formation. The multi-shelfrocker can rock at 5-20 cycles/min. The multi-shelf design can allow forproper rocking of all boxes (as opposed to stacking boxes on a singleshelf rocker). Rocker boxes can have a silicone rubber membrane with gasinflow/outflow circuit underneath (72-75% Nitrogen+21% O₂, 4-7% CO₂—CO₂can be used to maintain pH within a physiologic range). Oxygenation andpH of the media during spheroid formation can be determined by the gasinlet conditions. One purpose of the multi-shelf rocker can belarge-scale production of spheroids from freshly isolated orcryopreserved hepatocytes.

FIG. 4 c shows a spinner reservoir (e.g., a 2 liter volume spinnerreservoir) for extracorporeal use. FIG. 4 d schematically shows thespinner reservoir of FIG. 4 c. In some cases, the spinner reservoir caninclude a spin basket filter. The filter can rotate at about 30-200 rpm.In some cases, the filter can spin unidirectionally when in use. Thefilter can be designed such that bidirectional flow is allowed to unclogthe filter. The pore size for the filter mesh can be between 10-100microns. In some cases, the mesh has a pore size of 20-40 microns orsmaller. In some cases, the spinner reservoir can include a siliconerubber membrane that is gas permeable. The membrane can have a largesurface area for greater gas exchange which may support cell viabilityand biochemical activity at an increased cell density. Heparin and/orCoumadin can be used in both the spinner reservoir and the rockerreservoir at concentrations to balance anti-cogulation with spheroidsize. In some implementations, 1 IU/mL heparin and 1 μg/mL Coumadin areused. The spinner design can serve several purposes including tomaintain the spheroids in suspension and to allow continuous perfusionof the spheroid reservoir with minimal loss of spheroids from thereservoir. The cell filter inside of the spinner reservoir can becontinuously cleaned of cells by the continuous inflow and outflowdesign of the propeller shaft. This continuous action can minimize thebuild up of cells on the filter, avoids plugging of the filter, andminimizes the loss of cells from the reservoir. A high velocity inflowand low velocity out flow can be achieved by adjusting thecross-sectional area of the inflow and outflow areas.

In some cases, the spheroid reservoir can be a container (e.g., acylindrical container) having up of two compartments (e.g., a mixingchamber and a settling volume chamber) that can be separated from oneanother by, for example, a partition in the form of a fenestrated funnelas illustrated in FIGS. 15 a and 15 b. One or more stir bars can belocated in the mixing chamber. For example, a stir bar can be placed atthe center and base of the mixing chamber. This spinning action cancreate a vortex effect that pulls flow along with cells down through thespout of the funnel and thus can minimize the net loss of spheroids outof the mixing chamber. The fenestrated funnel can dampen the conversionof radial flow lines to axial flow lines and can allow the top volume ofthe reservoir to serve as a settling column. This dampening effect canbe enhanced by a vortex that can be created as flow crosses thefenestrated funnel and is drawn down through the spout of the funnel by,for example, a centrally located stir bar at the bottom of the mixingchamber. Continuous flow can enter the reservoir at the base within themixing chamber and can exit the reservoir through an outlet at thecenter and top of the container. In some cases, the reservoir can havean opening for a temperature probe and an opening for a sampling port.

A fenestrated funnel can be composed of a variety of materials such aspolypropylene, polyethylene, or polycarbonate. The material can be amaterial that is not susceptible to cell sticking. One purpose of thefenestrated funnel can be to create a settling column in the uppervolume of the reservoir while allowing a mixing chamber in the lowervolume of the reservoir. Studies can be performed to identify an optimalposition of the funnel spout. Locating the spout closer to the bottom ofthe mixing chamber can enhance the vortex effect and thus can beassociated with a reduced number of particles leaving the mixing chamberand entering the settling column and exiting the reservoir through itsoutlet. A distance less than 5 cm from the chamber bottom can beassociated with fewer particles existing, and the optimum height fromthe bottom can be about 1-2 cm above the bottom of the mixing chamber.Other specifications for the design of the fenestrated funnel reservoircan be as follows. The bioreactor height can be at least 24 cm tall(including the settling volume) (e.g., between 24 cm and 100 cm, between24 and 75 cm, or between 24 and 50 cm), the fenestrated funnel itselfcan be between 10 and 20 cm tall (e.g. about 16.5 cm tall), the angle ofthe funnel can be between about 50° and 70° (e.g., about 60°), the spoutdiameter can be between about 0.5 and 2 cm (e.g., about 1.25 cm of atleast about 1 cm in some cases), the fenestrations can be between about1 to 4 mm wide (e.g., at least 1 mm wide), the funnel geometry can bedetermined such that the area of the windowed openings are about equalto the cross-sectional area of the settling column, and the percent openarea of the fenestrated funnel can be no less than about 50 percent.

As described herein, a variety of studies were conducted usingmicrobeads and spheroids made from rat and pig hepatocytes. Thesestudies demonstrate that the settling column effect is able to separatespheroids measuring 60 microns or greater at a settling velocity of 1-2cm/min (0.018 to 0.034 cm/sec). Based on these flow parameters, thespheroid reservoir can be operated at a flow rate of 400 mL/min if itscylindrical inner diameter is 7 inches (approximately 180 mm).Naturally, the optimal conditions for the system may be influenced bythe number of spheroids in the reservoir and the diameter of thesespheroids since larger spheroids have a faster settling rate and couldtolerate a higher axial velocity within the settling column.

When using the design of the fenestrated funnel bioreactor, a stirringrate range of 30 to 200 RPM can be used. The contents can be well mixedwithout overly agitating the settling volume. The stir bar length can bebetween 2 and 15 cm (e.g., 8 cm). The overall bioreactor can becylindrical in shape. The stir bar and funnel can be axially alignedwith the center axis of the cylindrical bioreactor. The flow rate can be100 to 400 mL/min with 400 mL/min being optimal based on human liverfunction. The device can have a single inlet and a single outlet. A testor sampling port can be provided into the mixing chamber. In some cases,a temperature probe port can be provided. A 1 μm filter (or smaller) canbe included in the Bio-Artificial Liver circuit on the outlet of thebioreactor. This filter could be incorporated into the bioreactor bylocating it between the settling column and the outlet. The bioreactormust be made from biocompatible materials (USP class 6). A 1 um filter(or smaller) is included in the bio-artificial liver circuit on theoutlet of the bioreactor. This filter could be incorporated into thebioreactor by locating it between the settling column and the outlet. A1 um filter (or smaller) is included in the SRBAL circuit on the outletof the bioreactor. This filter can be incorporated into the bioreactorby locating it between the settling column and the outlet.

In some cases, the spheroid reservoir (e.g., the spheroid reservoirhaving a fenestrated funnel) can be made primarily from acrylic usingstandard machining operations. The funnel can be made from ABS using afluid deposition modeling rapid prototyping process. The entire devicecan be made using plastic molding processes (e.g., injection and/or blowmolds).

In some cases, a spheroid reservoir can be of a SRBAL device providedherein can have a design as shown in FIG. 16 or 17.

A SRBAL device provided herein can provide liver detoxification topatients in liver failure. Detoxification can be provided bymetabolically active hepatocytes (e.g., primary porcine hepatocytes)within the spheroid reservoir, as well as the charcoal column and resincolumn within the albumin perfusate. Metabolic activity of hepatocytesin the spheroid reservoir can be assessed by cell viability, oxygenconsumption, albumin production, and P450 activity of the primaryhepatocytes via a sample port in the reservoir. The device can bedesigned to function extracorporeally (e.g., at the bedside of thepatient with acute liver failure (ALF)). The device may serve as analternative to liver transplantation in the possibility of spontaneousrecovery of liver failure or as a bridge to liver transplantation ifliver failure is not reversible.

A SRBAL provided herein can provide a synergistic benefit by combiningalbumin dialysis with hepatocyte-based therapy. Modifications such asusing fresh rather than cryopreserved hepatocytes, increasing the doseof hepatocytes from 70 grams to either 200 grams or 400 grams,lengthening the duration of therapy to continuous perfusion, along withadded benefits of albumin dialysis, may also make BAL therapyefficacious in ALF. The SRBAL can also detoxify ammonia. One way toincrease ammonia detoxification is by enhancing the transcriptionfactors that regulate ureagenesis gene expression, such as HNF6. In somecases, the SRBAL therapy can have two-fold benefits by both providingessential hepatocellular functions, such as detoxification of ammonia tourea, while also reducing the SIRS response to acute liver injury.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 The SRBAL Improves Survival and Reduces ExtrahepaticManifestations in a Canine Model of D-Galactosamine-Induced FHF

Testing of the SRBAL was conducted on six dogs (15-20 kg wt.) underthree treatment conditions: Fresh Hepatocyte SRBAL (2×10¹⁰ fresh pighepatocytes), No Cell BAL (control), and No BAL (control). Afterinduction of isoflurane anesthesia, four probes were placed in the dogbrain to measure ICP, microdialysis fluid, temperature, and tissueoximetry. Dogs received D-galactosamine (2.0 g/kg) at t=0 hr; BALtherapy was initiated at t=24 hr. Dogs received a 5 mmole bolus of NH₄Clon day 1 (t=6 hr) and three 5 millimole boluses of NH₄Cl at t=28 hr,t=32 hr, t=36 hr to assess detoxification of NH₃ before and after onsetof liver failure. Arterial NH₃ samples were obtained immediately beforeand 60 minutes after NH₄Cl boluses. It was observed that dogs treatedwith the hepatocyte SRBAL group maintained normal intracranial pressuresand survived the experimental period, while all control animalsdeveloped intracranial hypertension and died from brain herniation(Table 1).

TABLE 1 Summery Table-Preliminary Studies of SRBAL as Treatment of ALFDogs Fresh No Cell BAL- No BAL- Hepatocyte Control Control EndpointUnits BAL n = 2 n = 2 n = 2 Survival hr EOT * 35, 41 36, 40 ALT (peak)U/L 6020, 8040 6520, 7300 8090, 7003 ICP (final peak) mmHg 20, 23 56, 6955, ** NH₃ (peak blood level) μmol/L 45, 65 101, 272 131, 179 BrainGlucose (final) mmol/L 5.2, 9.3 0.23, 0.42 0.34, **   Brain Lactate(final) mmol/L 2.8, 1.5 2.3, 8.9 5.8, **  Brain Glycerol (final) umol/L26.5, 39.4 117.2, 163.4 206.6, **   * end of therapy ** intracranialprobes not placed

Improved outcome in the hepatocyte treatment group occurred in thesetting of similar liver injury, based on peak serum ALT levels, in allthree groups. Herniation was associated with a sudden decline in braintissue oxygenation (<10 mmHg) and reduced cerebral blood flow. Brainglucose declined and brain lactate increased in control animalsreflecting cerebral ischemia with herniation. The marked rise in brainglycerol observed in control animals at the time of herniation wasfurther evidence of acute neuronal damage and was diagnostic of braindeath. In contrast, final brain levels of glucose, lactate, and glycerolwere normal in dogs treated with fresh hepatocytes in the SRBAL.

An added benefit of treatment with the SRBAL was improved detoxificationof NH₃. In fact, the response of ALF dogs to a 5 millimole bolus ofNH₄Cl during SRBAL treatment was similar to their response on day 1before onset of ALF (0.47±0.81 vs. 0.34±0.63 μmol/L/mmol, p=NS). Incontrast, the response of ALF dogs in the No Cell BAL group to the same5 millimole bolus of NH₄Cl was significantly elevated from baseline(3.47±1.77 vs. 0.63±0.34 μmol/L/mmol, p=0.025). Improved ammoniadetoxification by the SRBAL was associated with high viability ofporcine hepatocyte spheroids at the end of BAL therapy (viability>90%).Oxygen consumption by hepatocyte spheroids in the SRBAL also remainedstable, ranging from 0.94 to 0.88 mmol O₂/hr from start to end ofextracorporeal therapy.

Example 2 Treatment of ALF Dogs with the Hepatocyte SRBAL Contributes toSustained Normalization of the Systemic Inflammatory Response Syndrome(SIRS)

Treatment of ALF dogs with the hepatocyte SRBAL was associated withsustained normalization of the systemic inflammatory response syndrome(SIRS). Hallmarks of SIRS such as hypotension, oliguria, and respiratorydysfunction (FIG. 5 a) were observed in control dogs beginning 12 hoursafter administration of D-galactosamine. A rise in blood levels of TNFαwas observed in control dogs at 24 hours post D-galactosamine. Bloodlevels of TNFα rose steadily until expiration in the no BAL group. Asharp rise in TNFα was observed at the initiation of No cell BAL therapyand remained elevated in the range of the No BAL group up until braindeath. In contrast, normal levels of TNFα were measured in both dogstreated by the hepatocyte SRBAL (FIG. 5 b). These results indicate thatliver cells improve biocompatibility of the SRBAL and reduce the SIRSresponse of D-gal-induced ALF. The SIRS response of ALF was associatedwith a transient rise in LPS levels in the portal vein but not hepaticvein (FIG. 5 c), suggesting clearance of LPS by cells, such as Kupffercells, in the liver. However, this short burst in LPS could not fullyexplain the sustained SIRS response observed after administration ofhepatotoxin. These studies suggest the existence of other mediators ofSIRS of ALF besides LPS. Mechanistic studies are conducted to identifyendogenous mediators of the SIRS of ALF which may be cleared duringSRBAL therapy such as degradation productions of the extracellularmatrix.

Example 3 Functional TLR4 Signaling Assists in the Development of LiverPathology and SIRS after Toxic Exposure to Acetaminophen or D-gal/LPS

The role of TLR4 in ALF and SIRS was studied. FIG. 6 a shows that TLR4deficient (mutant) mice are protected from Dgal/LPS induced liver injurywhile wild-type mice develop severe necrosis of their liver withextensive TUNEL(+) apoptosis after exposure to Dgal/LPS. A similarprotective effect of TLR4 deficiency was also observed when wild-typeand TLR4 mutant mice were treated with acetaminophen (APAP) at 500mg/kg—a dose which is highly toxic with 100% mortality in wild-type mice(Table 2). Systemic inflammation was confirmed in wild-type mice afterboth APAP and Dgal/LPS by rising blood levels of TNFα and neutrophilinfiltration in their lungs at necropsy. In contrast, lung histology andsystemic levels of TNFα remained normal in TLR4 deficient mice. FIG. 6 bsuggests that hepatocyte death and the SIRS response to toxin-inducedliver injury is mediated by TLR4 expressed on Kupffer cells. TLR4deficient Kupffer cells may also explain the protective effects observedin TLR4 deficient mice after toxin exposure (FIG. 6 c). Consistent withthis observation, a significant reduction in the toxicity of Dgal/LPSwas observed in wild-type mice pretreated with gadolinium chloride(Table 2). Pretreatment with gadolinium chloride reduced the number ofKupffer cells in the liver by 48% (31±6 to 16±4 cells/HPF, p<0.001), andlowered peak ALT levels from 2458±2347 U/mL to 278±196 U/mL.

TABLE 2 Summary Table of Liver Injury TLR4 + (Wild type) vs. TLR4 −(Deficient) TUNEL Condition ALT (U/mL) (+cells/HPF) (n = 10/group) TLR4+TLR4− TLR4+ TLR4− APAP (18 hr) 9054 ± 4793   4810 ± 4045 * — — DGal/LPS(6 hr) 2458 ± 2347    125 ± 131 ** 177 ± 45  88 ± 41 * DGal/LPS/ 278 ±196 196 ± 127 — — Gadolinium (6 hr) ζ LPS alone (6 hr) 153 ± 69    46 ±16 * 4 ± 2   <1 * DGal alone (6 hr) 43 ± 9  54 ± 18 <1 <1 Saline (6 hr)27 ± 28 31 ± 14 <1 <1 * p value < 0.05 (vs. wild type, same condition)** p value < 0.001 (vs. wild type, same condition)

Further evidence supporting TLR4 as a signaling molecule in the SIRSresponse to ALF is illustrated in FIG. 7. FIGS. 7 a-b show the assay, aswell as the TLR4 receptor complex, used in the study. FIG. 7 cdemonstrates that serum obtained from critically ill mice, 6 hours afterAPAP 500 mg/kg, contains high levels of TLR4 agonist activity comparedto 0 mg/kg sham controls (p<0.001).

Example 4 To Determine the Optimal Operating Conditions of the SRBALBased on a Bioengineering Analysis of Factors Influencing SpheroidFormation, Biochemical Performance, Mass Transfer Across the BALMembrane, and Membrane Biocompatibility

1. Regulation of Spheroid Formation to Improve Biochemical Performanceof the SRBAL

In order to determine whether spheroid formation could be regulated toimprove biochemical performance of the SRBAL, spheroid formation bytraditional rotational technique and by a rocker technique described inU.S. Pat. No. 25,160,719 were first compared. Several advantages of therocker technique over the rotational technique were observed. Theseadvantages include a much faster rate of spheroid formation, bettercontrol of spheroid size, and greater percent of hepatocytesincorporation into spheroid aggregates by the rocker technique. After 24hours of rocking, most (85%) of inoculated hepatocytes had incorporatedinto well-formed spheroids of greater than 40 μm in diameter (FIG. 8).

At 48 hours, only 13% of cell particles measured less than 40 μm indiameter by Coulter counter measurement. The majority of rocker-formedaggregates (84%) ranged in diameter of 75-200 μm. In contrast, only 58%of hepatocytes were incorporated into spheroids under rotationalconditions at 24 hours. Most unattached hepatocytes appeared dead.Spheroid formation provided a protective benefit as greater than 95% ofall hepatocytes present in spheroids were viable at each time point upto 14 days (FIG. 9). Improved kinetics of spheroid formation and stablespheroid integrity by rocked technique may be associated with greaterbiochemical performance of spheroid hepatocytes compared to monolayercontrols.

Gene expression of rat hepatocytes over 14 days of rocked spheroidculture was profiled. A custom microarray of 242 liver-related genes wasdeveloped for this task. It was observed that the expression of 85% ofthese genes remained stable—less than a 2-fold increase or 50% decreasein expression—over 14 days in culture (FIG. 10). Expression increased2-fold or greater in 5% of genes; and decreased 50% in 10% of genes.Biochemical activity of rocked spheroid hepatocytes was also superior tospheroids formed by rotational technique and to hepatocytes cultured asmonolayers. These results suggest that spheroid formation can beregulated to improve biochemical performance of the SRBAL. Conditionsfor better spheroid formation include serum free medium supplementedwith insulin/transferrin/selenium at a rocker frequency of 15 cycles perminute, and a seeding density of 5×10⁶ viable hepatocytes per mL.Following 24 hours of rocking, newly formed spheroids can beconcentrated to a density of 1−2×10⁷ cells/mL if placed in the SRBALwith continuous oxygenation and supplementation with fresh medium.

2. Influence of Flow Conditions in its Extracapillary Space onBiochemical Performance of the SRBAL

The influence of flow conditions on performance of the SRBAL was studiedusing a four-pump bench-top apparatus as illustrated in FIG. 11.Variables considered in the optimization studies included hollow fibermembrane pore size (70 kD, 150 kD, 400 kD), pump configuration (F=0, 5,25, 50 mL/min), and albumin concentration in R2 (0.5 g %, 5.0 g %).These three variables accounted for a total of 24 conditions that weretested using three waste molecules (ammonia, conjugated & unconjugatedbirubin), pig albumin, and three immune molecules (TNFα, IgG, and IgM)as markers of permeability. In all studies, Reservoir #1 was initiallyfilled with pig plasma supplemented with each of these compounds.Reservoir #2 was primed with PBS supplemented with either 0.5 g % or 5.0g % bovine albumin. Tubing and cartridges were primed with solutionscorresponding to their side of the hollow fiber membrane. In addition,sieving coefficients of all membranes were determined by a standardizedmembrane technique using dextran polymers before and after permeabilitytesting. A rank sum analysis of all 24 conditions and 8 measures ofpermeability was performed to determine the best condition for operationof the SRBAL in the pre-clinical efficacy studies. The rank sum analysisdetermined that mass transfer of albumin (p<0.001), TNFα (p<0.001) andunconjugated bilirubin (p<0.01) was greatest under high flux usingeither 150 kD or 400 kD membrane. When permeability of IgG and IgM wasalso considered, the 150 kD membrane was considered superior due to itslower permeability to these potentially harmful antibodies.

3. Influence of the Concentration of Albumin in the BAL Medium onElimination of Polar and Non-Polar Toxins from the Patient Compartment

The influence of albumin concentration in the BAL medium (R2) onelimination of polar (ammonia, conjugated bilirubin) and non-polar(unconjugated bilirubin) toxins from the patient compartment (R1) weretested using the bench-top apparatus in FIG. 11. Slightly higher ratesof mass transfer were observed for all three molecules under conditionsof high (5.0 g %) vs. low (0.5 g %) albumin. The studies favor use ofstandard albumin dialysis conditions during SRBAL therapy.

4. Influence of Permeability of the BAL Membrane on Biocompatibility,Immuno-Protection, and Biochemical Performance of the SRBAL

The above studies demonstrated that mass transfer of albumin and wastemolecules was greatest using the either 150 kD or 400 kD membrane. The150 kD membrane can be used to limited transfer of larger andpotentially harmful molecules of IgG and IgM into R2.

Example 5 To Establish Efficacy of the SRBAL in a Preclinical Model ofALF by Demonstrating Improved Survival and a Reversal of HepaticEncephalopathy

1. Influence of the Dose of Freshly Isolated Porcine Hepatocytes in theReservoir Medium on Efficacy of the SRBAL

As reported in Table 1 above, studies of the SRBAL using 200 grams offresh porcine hepatocytes demonstrated a beneficial response in thetreatment of two dogs with D-gal-induced ALF. Validation studies usingfreshly formed spheroids or porcine hepatocytes at a dose of 200 gramsor 400 grams are described in Example 8.

Example 6 Benefit of Faster or Slower Rotational Speed in SpheroidReservoir

Two 1.5 L rocker boxes were inoculated with 1 liter of hepatocytesuspension (5×10⁶ cells/mL) and cultured for 24 hours (red line).Spheroids were divided and re-suspended in 2 liters of fresh media andinoculated into one of two SRBAL spin reservoirs. One reservoir wasagitated at 98 rpm (green line) and the other at 196 rpm (blue line) for22 hours. At the end of 22 hours, 20 mL of spheroid suspension wasremoved from each reservoir, pelleted, suspended in fresh media andinoculated into glass rocker plates. Samples for albumin production andBUN were taken at 4 hours and 24 hours. The results of this study areshown in FIG. 12.

These results demonstrate that faster spinner rates can lead to smallerspheroids, which can be advantageous under some conditions. Theseresults also demonstrate that slower spinner rates can lead to largerspheroids, which can settle faster in a settling column and can be lesslikely to exit the settling column. [Is this accurate?]

Example 7 Formation of Spheroids in Both Serum-Containing and Serum-FreeMedium

As shown in FIG. 13, spheroids can be formed both in medium that hasserum and in medium that is serum-free. There appears to be a benefit ofadding 10% calf serum and 10 mM L-carnitine to medium during spheroidformation. For example, 81% of hepatocytes were incorporated intospheroids when RPMI spheroid forming medium was supplemented with 10%FBS and 10 mM L-carnitine and a suspension of freshly isolated pighepatocytes was rocked at 0.25 Hz for 24 hours.

Example 8 To Establish Efficacy of the SRBAL in a Preclinical Model ofALF by Demonstrating Improved Survival and a Reversal of HepaticEncephalopathy

1. A Dose Response Study of the SRBAL Using Xenogeneic (Pig) Hepatocytes

The efficacy of the SRBAL in ALF is assessed in two sets of experiments(xenogeneic dose study, allogeneic study). Recipient model, BALconditions and endpoints for each of these two experiments aresummarized in Table 3. The Dose study is intended to determine theoptimal number of hepatocytes in the SRBAL and whether there is asynergism between albumin dialysis and hepatocytes in the perfusatecircuit. The allogeneic study is to determine if there is additionalbenefit to using hepatocytes from the same species as the patient. Theperfusate circuit in both studies includes physiological levels ofalbumin (5 g %) along with charcoal and resin columns used commerciallyin MARS™ albumin dialysis. The SRBAL can be operated at conditions ofhigh flux (50 mL/min) using the 150 kD or 400 kD MWCO modifiedpolysulfone hollow fiber membrane. Dose studies use fresh pighepatocytes ranging from 0 to 400 grams. The 0 gram group is comparableto standard albumin dialysis. A no BAL control group is included toassess the baseline inflammatory response of ALF dogs in the absence ofan extracorporeal device, which may elicit an inflammatory response assuggested in FIG. 5. The allogeneic study uses the same dose ofhepatocytes found to be most efficacious in the dose study butallogeneic. Survival duration is the primary endpoint of both the dosestudy and allogeneic study. Other secondary endpoints listed in Table 3are carefully monitored and reported.

TABLE 3 Summary of Studies Test Efficacy of SRBAL Xenogeneic AllogeneicDose Study Study (n = 10/group) (n = 5/group) Recipient ModelD-galactosamine-Induced ALF dog x x BAL Conditions ** Pig Spheroids 400g + 5 g % Albumin x Pig Spheroids 200 g + 5 g % Albumin x No Spheroids 0g + 5 % Albumin x Dog Spheroids ? g + 5 g % Albumin x No BAL x EndpointsSurvival (hours) x ^(a) x ^(a) Intracranial pressure (mmHg) x x ^(b)Cerebral microdialysis (glycerol, glucose, x x ^(b) lactate, ammonia)Pulmonary Function (arterial pO2, pCO2) x x Renal Function (serum Cr,urine output) x x SIRS markers (TNFα, IL6) x x Liver Injury (ALT,ammonia, glucose, x x lactate) Hemodynamics (MAP, HR) & Temperature x xHepatocyte Viability (pre vs. post) x x Biochemical Performance of SRBAL^(b) x x Autopsy (liver, lungs, kidney, brain) x x ^(a) primary endpoint^(b) assessed by ureagenesis, albumin production (pre vs. post), O₂consumption (during) ? Optimal dose of allogeneic hepatocyte spheroidswill depend on outcome of the Xenogeneic Dose Study

Efficacy testing of the SRBAL is conducted in the canine D-galactosaminemodel of ALF (see, e.g., Sielaff T D, Nyberg S, Amiot B, Hu M, Peshwa M,Wu F, Hu W-S, et al. Application of a bioartificial liver (BAL) in a newmodel of acute fulminant hepatitis. Surgical Forum 1993; 44:61-63;Nyberg S, Cerra F, Gruetter R. Brain lactate by magnetic resonancespectroscopy during fulminant hepatic failure in the dog. LiverTransplantation and Surgery 1997; 4:158-165). This animal model isselected because of its similarity (hepatic encephalopathy progressingto cerebral edema and brain death, pulmonary edema, hepatorenalsyndrome) to clinical ALF. Dogs are instrumented to monitor changes inhemodynamics (pulmonary function, renal function and brain physiology)during development of liver failure (and reversal by SRBAL). Endpointsused for monitoring efficacy of the SRBAL include: 1) survival time, 2)intracranial pressure and cerebral microdialysis as measures of cerebraledema, 3) pulmonary and renal function as other extrahepaticmanifestations of ALF, 4) hemodynamics of the animal, 5) levels of TNFαand IL6 as markers of SIRS, and 6) ALT/ammonia/glucose/lactate asmarkers of liver injury and function. Hepatocyte viability andbiochemical performance of porcine hepatocyte spheroids is assessedbefore, during and after treatment as an indirect measurement of immuneprotection. Finally, an autopsy is performed on each animal to carefullyexamine brain, lungs and kidney as well as consistency of liver damagein study animals. A direct correlation may be seen between liverfailure, toxin accumulation and extrahepatic manifestations of liverfailure, and improvement if this sequence of events is reversed by SRBALtherapy. There may be a synergism between hepatocytes and albumindialysis.

As in Examples 1 and 2 provided herein, dogs are intubated andinstrumented under isoflurane anesthesia before administration ofD-galactosamine at T=0 hours. Perfusion of the extracorporeal circuit isinitiated at T=12 hours after administration of D-galactosamine. TheSRBAL has been under in vitro conditions for at least 24 hours to insureproper formation of spheroids and maximum biochemical performance at theinitiation of treatment. Ammonium chloride bolus is administered at 6hour, 18 hour and continuously after 24 hours to assess ammoniadetoxification activity before liver failure, after liver failure, andduring SRBAL therapy.

The spheroid reservoir design is suited for these studies since thedesign allows continuous sampling of hepatocytes for viability andmedium for measurement of oxygen consumption before, during, and aftertreatment. Biochemical performance of SRBAL is measured at baseline(before treatment) and for 24-hours after completion of therapy. Animalsare treated up to 48 hours.

A power analysis is performed using data from Table 1 above to establishgroup size for efficacy testing in this study. According to Table 1,control ALF dogs had a survival of (35, 36, 40, 41 hours, mean—38 hours,st dev—3 hours). Therefore, assuming an alpha of 0.05 and 80% power, thefollowing differences in mean survival between treatment and controlgroups are significant: 6.3 hours if n=5; 4.5 hours if n=8; and 3.0hours if n=10. A more conservative mean survival difference of 3 hoursand n=10 dogs per group is chosen since Table 1 was limited to only 4control dogs. These assumptions are valid based on the studies in theExamples.

40 dogs are treated. There is a synergistic effect of albumindialysis+spheroid hepatocyte therapy at both low (200 gram) and high(400 gram) doses of pig hepatocytes. The longest survivors are in the400 gram pig spheroid treatment group. Therapy with all three treatments(AD alone, AD+200 gm, AD+400 gm) may be associated with improvement insecondary endpoints such as lower intracranial pressure, normalizedcerebral microdialysis values, improved pulmonary and renal function,and reduced systemic inflammation based on lower values of TNFα and IL6.

If the dose of 2.0 gm/kg D-galactosamine is too high and results inlethal ALF beyond the level that can be stabilized by SRBAL therapy, alower dose of 1.0-1.5 gm/kg D-galactosamine may be used instead. TheSRBAL can be operated at conditions of high flux (50 mL/min) using the150 kD MWCO modified polysulfone hollow fiber membrane. If hepatocyteviability is high at the end of therapy but the benefit of therapy isbelow expectation (non-significant survival benefit), the system canthen be revised to the 400 kD MWCO. If such changes are required, thenadditional animals are studied such that n=10 per group.

2. The Therapeutic Benefit of Allogeneic Hepatocytes in the SRBAL

The same study as #1 is performed except that dog hepatocytes are usedin the SRBAL device. The techniques for isolating hepatocytes from dogsand pigs are identical. The dose of hepatocytes loaded in the SRBALequals the dose found to be most efficacious in #1. 5 dogs are studiedin order to determine if there is an incremental benefit of usinghepatocytes from an allogeneic source in the SRBAL.

Results similar to those in #1 may be achieved with allogeneic andxenogeneic hepatocytes at the same dose. Since immune-mediatedcomplications can be less likely with an allogeneic source ofhepatocytes, hepatocyte viability may be higher when examining posttreatment. The 400 kD hollow fiber membrane is used in the allogeneicstudy which may improve the efficacy of therapy—longer survivalduration.

Example 9 Determine the Role of a Cell-Based Bioartificial Liver inQuenching the Systemic Inflammatory Response Syndrome of Acute LiverFailure (sirs of alf)

1. Screen Portal Vein, Hepatic Vein, and Arterial Blood of BAL Treatedand Untreated ALF Dogs for Evidence of TLR4 Agonist Activity

Dogs from Example 1 provide blood samples that are screened formeasurement of TLR4 agonist activity before and every six hours duringonset of D-gal-induced ALF. These blood samples are obtained from theportal vein, hepatic vein, and femoral artery of SRBAL treated dogs anduntreated control dogs. Two weeks prior to induction of ALF, dogsundergo a surgical procedure to place two BARD™ port-style catheters inthe hepatic vein and portal vein. This procedure involves a laparotomyunder general anesthesia to place the internal end of the catheter intothe appropriate vein. The external portion of the catheter is tunneledto a site on the dog's back for subcutaneous placement of the port. Thissurgical procedure is more reliable than catheter placement bypercutaneous radiological technique. A two week interval betweencatheter placement and induction of liver failure allows full recoveryof the dog without interference from the inflammatory response oflaparatomy. Femoral artery catheters are placed by percutaneoustechnique the day of D-galactosamine infusion. Ports are also accessedby Huber needle at that time.

TLR4 agonist activity are measured by an in vitro assay usingHEK293/Luc(+)/TLR4(+) cells (see, e.g., Akira S, Takeda K. Toll-likereceptor signaling. Nat Rev Immunol 2004.; 4:499-511). This assay, aswell as the TLR4 receptor complex, is illustrated in FIGS. 7 a-b.Luciferase reporter gene signal are quantified by TD-20/20 luminometer(Turner Designs, Sunnyvale, Calif.). Activation of NFκβ is reported as aratio of firefly luciferase activity to the constitutively expressedRenilla luciferase internal control from a mean of triplicate wells(see, e.g., Brunn G, Bngm M, Johnson G, Platt J. Conditional signalingby Toll-like receptor. FASEB J 2005; 19:872-4).

The development of acute liver failure may be associated with a SIRSresponse and a rise in TLR4 agonist activity in systemic arterial bloodsamples. A rise in TLR4 agonist activity may be seen in blood sampledfrom portal vein and hepatic vein. There may be differences in profilesof TLR4 agonist activity depending on whether the gut or the liver isthe primary source of TLR4 activators. TLR4 activity may show an earlyrise in portal vein followed by a late sustained rise in hepatic veinblood as liver injury becomes established. SRBAL therapy may beassociated with normalization of TLR4 agonist activity sincenormalization of TNFα—a sensitive marker of systemic activation—may beobserved during SRBAL therapy.

2. ECM Components Released During Liver Injury are Mediators of Sirs andthese Potential Mediators of Sirs are Cleared During Therapy with theSRBAL

Blood samples from portal vein, hepatic vein, and femoral arteryobtained from ALF dogs of Example 8 (with or without SRBAL therapy) arescreened for presence of TLR4 agonist activity using ourHEK293/Luc(+)/TLR4(+) cell in vitro assay. Samples found to havepositive TLR4 agonist activity are further analyzed to determine if thisactivity is related to the presence of LPS, heparan sulfate, and/orhyaluronic acid. These studies are conducted sequentially. The firststep is to remove LPS and retest samples with and without LPS removal.The second step is to remove heparan sulfate and retest samples with andwithout heparan sulfate removal. The third step is to remove hyaluronicacid and retest samples with and without hyaluronic acid removal. Adecline in TLR4 agonist activity after any of these three manipulationsconfirms the presence of that molecule (LPS, heparin sulfate, orhyaluronic acid) in the sample of dog blood, and more importantly,implicates that molecule as a mediator of the SIRS response in ALF.Removal of LPS is performed by incubating the sample in polymixin B, apositively charged detergent which binds LPS specifically (see, e.g.,Rifkind D. Studies on the interaction between endotoxin and polymyxin B.J Infectious Dis 1967; 117:433-438). Removal of heparan sulfate isperformed by incubating the sample in recombinant heparanase (see, e.g.,Brunn G, Bngm M, Johnson G, Platt J. Conditional signaling by Toll-likereceptor. FASEB J 2005; 19:872-4). Removal of hyaluronic acid areperformed by incubating the sample in bovine testes hyaluronidase (SigmaChemicals) (see, e.g., Termeer C C, Hennies J, Voith U, Ahrens T, WeissJ M, Prehm P, Simon J C. Oligosaccharides of hyaluronan are potentactivators of dendritic cells. J Immunol 2000; 165:1863-1870). Allmaterials used in cell culture are certified endotoxin free or tested bythe Limulus amebocyte lysate assay gel clot method (Seikagaku, FalmouthMass.) to assure absence of detectable endotoxin (<0.1 ng/mL).

It may be shown that the SIRS of ALF is mediated, at least in part, byendogenous molecules such as heparan sulfate or hyaluronic acid.

Serum obtained in the course of ALF may contain matrix metalloproteaseswhich cleave extracellular matrix proteins and potentiate TLR4activation by endogenous or microbial ligands such as LPS. To testwhether ALF serum contains these enzymes, HEK/Luc(+)/TLR4(+) cells arecultured on extracellular matrix-coated plates in the presence of ALFserum. Six hours later increasing amounts of LPS are added to separatewells of the cultured cells. Potentiation of TLR4 signaling by ALF serumis detected by a left-ward shift of the LPS-TLR4 activationdose-response curve, and indicates that ALF serum contains an indirectactivator or potentiator of TLR4 signaling. If LPS, heparan sulfate, andhyaluronic acid are not found in ALF dog serum, then other potentialcandidate molecules are considered. Other candidates include lauricacid, fibrinogen, fibronectin, heat shock protein, and β-defensin.Heparan sulfate and hyaluronic acid are leading candidates.

3. The Influence of Hepatocytes, Kupffer Cells, and Stellate Cells ofthe Liver on the Circulating Levels of ECM Components in ALF and theRoles of these Cells in Regulating the SIRS of Acute Liver Failure

This experiment includes a series of in vitro studies to determine therole of liver cells in regulating the SIRS response of ALF. Plasmasamples with TLR4 agonist activity obtained from control ALF dogs (noBAL group) of Example 8 are studied. Portal vein, hepatic vein andfemoral artery samples are considered. TLR4 agonist activity aredetermined in these samples using a HEK293/Luc(+)/TLR4(+) cell in vitroassay (see, e.g., Brunn G, Bngm M, Johnson G, Platt J. Conditionalsignaling by Toll-like receptor. FASEB J 2005; 19:872-4). Rat livercells are used in the studies (see, e.g., Seglen P. Preparation ofisolated rat liver cells. Methods in Cell Biology 1976; 13:29-83), andthe results are verified using porcine hepatocytes. Crude preps of livercells (85-90% hepatocytes, 5-10% Kupffer cells, stellate cells, etc.)are used in initial studies, followed by enriched preps of hepatocytes,Kupffer cells, or stellate cells in subsequent studies. Primaryhepatocytes are enriched by selective culture of crude preps inarginine-free medium available from Sigma Chemical (see, e.g., Leffert HL, Paul D. Studies on primary cultures of differentiated fetal livercells. J Cell Biol 1972; 52:559-568; Spiegelberg T, Bishop J O.Tissue-specific gene expression in mouse hepatocytes cultured ingrowth-restricting medium. Mol Cell Biol 1988; 8:3338-3344). Kupffercells are isolated by a four step technique employing enzymatictreatment, density gradient centrifugation, centrifugal elutriation, andselective adherence (see, e.g., Valatas V, Xidakis C, Roumpai H, KoliosG, Kouroumalis E. Isolation of rat Kupffer cells: a combined methodologyfor highly purified primary cultures. Cell Biol International 2003;27:67-73). Stellate cells are isolated by density gradientcentrifugation using arabinogalactan (see, e.g., Ramm G. Isolation andculture of rat hepatic stellate cells. J Gastroenterol Hepatol 1998;13:846-851). Enriched cell populations (1×10⁶ cells/mL) are incubated at37° C. for 24 hours in a serum free medium of RPMI, supplemented withinsulin/transferring/selenium (ITS, Sigma Chemical) and 0.5 vol % dogserum sample. Dog serum samples (0.5 vol %) are also incubated at 37° C.for 24 hours in a control condition of serum free medium without cells.TLR4 agonist activity are measured in both cell and no-cell (control)conditions to determine the effect of liver cell exposure on TLR4agonist activity. TNFα levels are measured in all Kupffer cell culturesand any samples that show a paradoxical increase in TLR4 agonistactivity.

A decline in TLR4 agonist activity may be seen after incubation of dogsample in crude and pure cultures of primary hepatocytes due tometabolic degradation of mediators of the SIRS response. A reduction inTLR4 agonist activity may also be seen after incubation in cultures ofpure Kupffer cells or pure stellate cells.

If the 0.5 vol % concentration of “toxic” sample is large enough tostimulate a TLR4 agonist response in HEK293/Luc(+)/TLR4(+) cell, higherconcentrations of toxic dog serum are tested (1-10 vol %). If ALF dogserum contains matrix metaloproteases, techniques of LPS titration areemployed to rule out contamination in this experiment if indicated. Ifsmall numbers of dendritic cells, known to express TLR4 and which arepresent in the spheroid aggregates, participate in the quenching of SIRSof ALF, the role of dendritic cells in the SRBAL are considered if thebeneficial results of SRBAL therapy cannot fully explained by removal ofhepatocytes, Kupffer cells, and stellate cells.

Example 10 Determine the Role of Hepatocyte Nuclear Factor 6 (hnf6) inMaintaining Ureagenesis in a Spheroid Reservoir Bioartificial Liver

1. A Recombinant Adenoviral Vector to Induce Stable Expression of HNF6in Rat Hepatocyte Spheroids

A CMV-GFP—HNF6 fusion expressing adenovirus plasmid (AdCMV-GFP-HNF6) isdesigned based on a cDNA clone containing the entire protein codingsequence of rat Hnf6 generated by polymerase chain reactionamplification using rat hepatocyte cDNA. To control adverse effects ofadenovirus infections, a GFP containing adenovirus plasmid (AdCMV-GFP),without Hnf6, is also be prepared. Rats are infected with stepwiseincremental concentrations of AdCMV-GFP-HNF6 until the optimalconcentration of adenovirus is found which successfully expressesHNF6-GFP in the majority of hepatocytes. Once this has been established,hepatocytes are isolated from uninfected, control infected (AdCMV-GFP)and AdCMV-GFP-HNF6 from male Sprague-Dawley rats (Harlan, Indianapolis,Ind.) by a two-step perfusion method (see, e.g., Seglen P. Preparationof isolated rat liver cells. Methods in Cell Biology 1976; 13:29-83).Only harvests yielding hepatocytes with a viability of at least 90%, asdetermined by trypan-blue dye exclusion, are used for subsequenthepatocyte spheroid cultures. Spheroid hepatocyte cultures are preparedby incubation of freshly isolated cells in siliconized 20 mL glassculture plates at a cell density of 1×10⁶ cells/mL under rockedconditions (see, e.g., Brophy C, Luebke-Wheeler J, Amiot B, Remmel R,Rinaldo P, Nyberg S. Rat hepatocyte spheroids formed by rocked techniquemaintain differentiated hepatocyte expression and function. Hepatology,2009, 49:578-86). Samples are harvested 1, 2, 7 and 14 days forsubsequent experiments. Changes in HNF6 expression are monitored bygreen fluorescence microscopy due to the fusion of green fluorescentprotein with HNF6. Flow cytometry is used to determine the percentage ofhepatocytes stably expressing GFP-HNF6. Briefly, spheroids fromadenovirus treated and untreated cultures are trypsinized andimmediately analyzed to determine the ratio of green fluorescent cellscompared to total cell number. Changes in Hnf6 expression due toadenoviral HNF6 infections are also verified by reverse transcriptionpolymerase chain reaction (qRT-PCR) (see, e.g., Brophy C, Luebke-WheelerJ, Amiot B, Remmel R, Rinaldo P, Nyberg S. Rat hepatocyte spheroidsformed by rocked technique maintain differentiated hepatocyte expressionand function. Hepatology, 2009, 49:578-86). In addition, changes in HNF6protein are monitored by Western blot analyses using an HNF6 antibodyfrom the multiple rat liver infected with either the control or HNF6expressing adenoviruses compared to uninfected cultures from isolatedadult rat livers.

Hnf6 expression may be induced and maintained in rat hepatocytespheroids since the expression of Hnf6 is under direct control of theCMV promoter. Hnf6 expression may be detected by quantitative qRT-PCRanalyses of Ad-CMV-HNF6 expressing hepatocytes and not by Ad-CMVinfected hepatocyte spheroids which could lose expression of Hnf6 over14 days in culture. Increases in HNF6 protein, determined by Westernblot analyses, may follow increases in Hnf6 gene expression.

If it is difficult to obtain HNF6 expressing cells by tail veininjection of an HNF6 expressing adenovirus, hepatocytes are directlyinfected under in vitro suspension culture conditions. Alternatively,HNF6 is re-expressed in rat hepatocyte spheroid cultures directly bytransiently transfecting CMV-HNF6 expressing plasmid into suspensions ofprimary rat hepatocytes.

2. The Effect of Overexpression of HNF6 on Expression of Urea CycleGenes and Their Function in Rocked Spheroid Culture

Rat hepatocyte spheroid cultures from uninfected, Ad-CMV infected (ascontrols) and Ad-CMV-HNF6 infected rat livers are performed as describedabove in #1. Initially, qRT-PCR analyses of all six ureagenesis genesare done to determine the effects of HNF6 on ureagenesis geneexpression. In order to detect biochemical performance of the hepatocytespheroids, culture media are spiked with ammonia sulfate anddetoxification rates are measured. The conversion of heavy ammonia(N¹⁵D₃) to heavy urea is used to detect complete urea cycle activity.

Cells which express HNF6 may have improved ammonia detoxificationabilities compared to untreated or control infected cultures which donot express HNF6. Changes in ureagenesis function may be due toincreases in the expression levels of ureagenesis genes.

If forced expression of HNF6 is sufficient to induce changes inureagenesis gene expression and function, other liver transcriptionfactors are studied to determine if additional regulatory factors arelost in hepatocyte spheroids. This is accomplished by qRT-PCR analysesof the expression of other hepatocyte transcription factors that areinvolved in the regulation of ureagenesis gene expression.

3. HNF6 can Regulate Cps1 Expression

Both Hnf6 and Cps1 expression is down regulating in hepatocyte spheroidcultures. This study involves a combination of transient transfectionand chromatin immunoprecipitation (ChIP) analyses. For transienttransfection analyses, a series of promoter constructs containingportions of the Cps1 upstream regulatory sequences driving theexpression of a luciferase reporter gene. An HNF6 expression plasmid isconstructed which allows for high levels of expression from thecytomegalovirus promoter (CMV) when transfected into tissue culturecells. These expression plasmids are introduced into NIH3T3 cells alongwith different fragments of the Cps1 promoter driving luciferase. HNF6is not normally expressed in NIH3T3 cells so these cells are used todetermine whether HNF6 transactivates expression via the Cps1 promoterby measuring relative luciferase levels. A comparison of the differentpromoter fragments to respond to HNF6 transactivation allows for mappingwhere HNF6 acts on the Cps1 promoter/enhancer. This information mayindicate where to focus initial analyses of direct HNF6 regulatoryregions. A direct in vivo relationship may be identified between HNF6and Cps1. To accomplish this adult rat livers and hearts (as a negativecontrol since they do not express HNF6) are isolated and fixed with 1%formaldehyde, cells dissociated using a homogenizer, and chromatin aresheared to approximately 500 bp by sonication. Immunoprecipitation ofchromatin is performed using the Upstate ChIP Assay Kit (Upstate#17-295) following the manufacturer's instructions with anti-HNF6 (SantaCruz, H-100) or anti-Pesl antibodies. Primers are designed to allow PCRamplification of regions of chromatin that flank a known HNF6 bindingsite in Cyp7a1, any predicted HNF6 binding sites in Cps1, and a fragmentof RNA Pol2, which lacks an HNF6 binding site as a negative control.

HNF6 may be capable of regulating ureagenesis functions by regulatingthe expression of ureagenesis genes, such as Cps1, in hepatocytespheroids, and HNF6 can transactivate Cps1 gene expression via upstreamCps1 regulatory regions using transient transfections assays. HNF6 mayindirectly regulate the expression of Cps1, and HNF6 may do this throughthe direct regulation of other transcription factors that directlyregulate Cps1 expression. Evidence of in vivo binding of HNF6 may not befound by ChIP analyses. HNF6 may directly exert its regulatory effectson Cps1 expression, and ChIP analyses may allow for identification ofpotentially novel HNF6 regulatory sequences.

If HNF6 is not sufficient to regulate the expression of Cps1 in NIH3T3cells, this may be due to the absence of additional liver transcriptionfactors required to transactivate the Cps1 promoter/enhancer in vitro.The transient transfection assay is performed in a hepatoma cell linessuch as HepG2 which does express most of the hepatic transcriptionfactors, albeit at lower levels then found in endogenous liver tissue.

Example 11 Spheroid Formation

With regards to conditions for spheroid formation, studies wereperformed using hepatocytes at three cell densities (2, 5, 10×10⁶cells/mL) cultured in medium supplemented with 10% fetal bovine serum.Conditions for spheroid formation that were varied included the rockerrate (8 vs 10 cycles/min) and oxygen tension within the spheroid formingmedia (low pO₂ 50-60 mm Hg and high pO₂ about 300 mm Hg). Based on theseconditions, a larger number of spheroids were formed at the slowerrocking speed and lower pO₂ environment in the rocking container. Forexample, 95% of the initial inoculant of pig hepatocytes formedspheroids of 40 microns or greater at rocking rate of 8 cycles perminute and low pO₂. In contrast, only 86% of pig hepatocytes formedspheroids of 40 micron diameter or greater when rocked at 10 cycles/minin a high pO₂ environment. The other two combinations of conditions wereassociated with 90% spheroid formation. Of note, low pO₂ environmentshould not lead to hypoxic conditions in which oxygen supply does notmeet the cellular demand for oxygen. Once the supply/demand condition ismet, it appears that increasing oxygen tension has a toxic effect duringspheroid formation and later during rocked spheroid culture.

Hepatocytes were isolated from pig liver, and spheroids were formed fromthese cells by rocker technique at low pO₂ 50-60 mm Hg and 8 cycles perminute rocking frequency using the multi-tray rocker shown in FIG. 4 b.FIG. 20 demonstrates a high efficiency of spheroid formation with93.7±2.4% of hepatocytes formed into spheroids after 24 hours and93.2±1.9% formed into spheroids after 48 hours under these conditions.The total volume of cells in the system also remained relativelyconstant over this 48 hour interval of spheroid formation as shown inFIG. 19. After 48 hours, formed spheroids were then placed into thefenestrated funnel SRBAL bioreactor shown schematically in FIG. 15 a andphotographically in FIG. 15 b. These spheroids were perfusedcontinuously with culture medium for 24 hours. FIG. 18 summarizes thesignificant rate of oxygen consumption by hepatocytes during this 72hour time frame. Of note, oxygen consumption was determined by syringetechnique during the first 48 hours, and a direct measure of thedifference between inflow and outflow oxygen tension was used todetermine oxygen consumption while in the SRBAL during the final 72hours. The difference in techniques explains the difference in values ofoxygen consumption determined on Day 2 (92.6 μmolO₂/min) and SRBAL t=0hr (42.3 μmolO₂/min) in FIG. 18. Both measurements were taken from thesame population of spheroids less than 2 hours apart.

The physical properties of SRBAL designs were tested using a mixture ofsynthetic microbeads that approximate the same size and densitydistribution of hepatocyte spheroid preparations. In these studies,microbeads were added to the reservoir. The composition of microbeads inthe reservoir and in the outflow line were determined from samples ofeach using a Coulter counter. The fenestrated funnel design exhibitedmore effective retention of microbeads in the reservoir with the fewestnumber of particles exiting in the outflow line. In the experiment shownin FIG. 21, no particles of >100 μm in diameter were observed to exitthe reservoir.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A bioartificial liver device comprising a reservoir chamberconfigured to house hepatocyte spheroids, wherein said reservoir chambercomprises a mixing chamber and a settling volume chamber, wherein saidmixing chamber is separated from said settling volume chamber by afunnel, and wherein said mixing chamber is in fluid communication withsaid settling volume chamber via at least one opening defined in saidfunnel.
 2. The bioartificial liver device of claim 1, wherein saidfunnel is a fenestrated funnel.
 3. The bioartificial liver device ofclaim 1, wherein said fenestrated funnel comprises openings between 100μm and 5 mm in diameter.
 4. The bioartificial liver device of claim 1,wherein said reservoir chamber comprises a magnetic stir bar within saidmixing chamber.
 5. The bioartificial liver device of claim 1, whereinsaid mixing chamber comprises an inlet port.
 6. The bioartificial liverdevice of claim 1, wherein said settling volume chamber comprises anoutlet port.
 7. The bioartificial liver device of claim 1, wherein saidfunnel comprises more than 25 openings.
 8. The bioartificial liverdevice of claim 1, wherein said openings are between 100 μm and 5 mm indiameter.
 9. The bioartificial liver device of claim 1, wherein saidreservoir chamber comprises a sampling port.
 10. The bioartificial liverdevice of claim 1, wherein said reservoir chamber comprises temperatureprobe.
 11. A bioartificial liver device, comprising: a plurality of cellcontainers for forming hepatocyte spheroids; a multi-shelf rockingdevice configured to rock said plurality of cell containers; and areservoir chamber configured to house said hepatocyte spheroids afterbeing formed in said plurality of cell containers.
 12. The bioartificialliver device of claim 11, further comprising an albumin dialysis system.13. The bioartificial liver device of claim 12, wherein said albumindialysis system comprises a blood separation cartridge, a charcoalcolumn, a resin column, and a dialysis membrane.
 14. The bioartificialliver device of claim 12, wherein said albumin dialysis system comprisesa pre-dilution circuit.
 15. The bioartificial liver device of claim 11,wherein the multi-shelf rocking device is configured to rock at about5-20 cycles/min.
 16. The bioartificial liver device of claim 11, whereinthe multi-shelf rocking device comprises one or more rocker boxes havinga membrane for gas inflow/outflow.
 17. The bioartificial liver device ofclaim 11, wherein said reservoir chamber comprises a mixing device tomaintain hepatocyte spheroids in suspension.
 18. The bioartificial liverdevice of claim 11, wherein said reservoir chamber comprises a screen ormesh configured to allow bi-directional fluid flow.
 19. A bioartificialliver device comprising a reservoir chamber configured to househepatocyte spheroids, wherein said reservoir chamber comprises a screenor mesh configured to allow bi-directional fluid flow.
 20. Thebioartificial liver device of claim 19 further comprising an albumindialysis system.
 21. The bioartificial liver device of claim 20, whereinsaid albumin dialysis system comprises a blood separation cartridge, acharcoal column, a resin column, and a dialysis membrane.
 22. Thebioartificial liver device of claim 20, wherein said albumin dialysissystem comprises a pre-dilution circuit.
 23. The bioartificial liverdevice of claim 19, wherein said screen or mesh has a pore size of about20-40 microns or smaller.
 24. The bioartificial liver device of claim 19further comprising a multi-shelf rocking device configured to rock aplurality cell containers to form hepatocyte spheroids.
 25. Thebioartificial liver device of claim 19, wherein said reservoir chamberfurther comprising a mixing device configured to maintain hepatocytespheroids in suspension.
 26. The bioartificial liver device of claim 19,wherein said reservoir chamber further comprising a gas permeablemembrane to facilitate gas exchange.
 27. A bioartificial liver device,comprising: an albumin dialysis system; and a reservoir chamber in fluidcommunication with said albumin dialysis system, said reservoir chamberbeing configured to house hepatocyte spheroids.
 28. The bioartificialliver device of claim 27, wherein said albumin dialysis system comprisesa blood separation cartridge, a charcoal column, a resin column, and adialysis membrane.
 29. The bioartificial liver device of claim 27,wherein said albumin dialysis system comprises a pre-dilution circuit.30. The bioartificial liver device of claim 27, further comprising amulti-shelf rocking device configured to rock a plurality of cellcontainers to form hepatocyte spheroids.
 31. The bioartificial liverdevice of claim 27, wherein said reservoir chamber comprises a mixingdevice to maintain hepatocyte spheroids in suspension.
 32. Thebioartificial liver device of claim 27, wherein said reservoir chamberis configured to allow bi-directional fluid flow.
 33. The bioartificialliver device of claim 27, wherein said reservoir chamber comprises a gaspermeable membrane to facilitate gas exchange.
 34. The bioartificialliver device of claim 27, further comprising a controller to stabilizefluid volume in said reservoir chamber.